Microscope apparatus and cell culture apparatus

ABSTRACT

An imaging section of a microscope apparatus captures a plurality of microscope images each having the focal position which differs in the field being same with a light flux having passed through a microscopic optical system. A region separating section separates a cellular region from a non-cellular region by using the plurality of the microscope images. A focusing position calculating section finds a focusing position in a target pixel included in the cellular region based on a brightness change in the position being same in the plurality of the microscope images. A three dimensional information generating section generates three dimensional information of a cultured cell based on a position of the cellular region and the focusing position in the target pixel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of. U.S. application Ser. No.12/923,004, filed on Aug. 27, 2010, which is a Continuation Under 35U.S.C. § 111(a), of PCT International Application No. PCT/JP2009/000308,filed Jan. 27, 2009, which claims the benefit of priority from JapanesePatent Application No. 2008-047270, filed on Feb. 28, 2008, the entirecontents of which are incorporated herein by reference.

BACKGROUND

1. Field

The present application relates to a microscope apparatus and a cellculture apparatus.

2. Description of the Related Art

Until now, in order to analyze structures and functions of a cell, threedimensional information of the cell has been acquired by an observationusing a microscope. As an example, Japanese Unexamined PatentApplication Publication No. 2006-23476 discloses a configuration foracquiring three dimensional information by scanning a cell with aconfocal laser scanning microscope.

However, when acquiring three dimensional information of a cell using aconfocal laser scanning microscope as described above, the cell isdamaged by the irradiation of excited light for a fluorescenceobservation, dyeing, etc., and therefore, there is room for improvement.In particular, the field of regenerative medicine is based on thepremise that the cell cultured in vitro is implanted into a human body,and therefore, it is strongly required to acquire three dimensionalinformation of a cell while suppressing the damage to the cell as muchas possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elevation view of an outline of a cell culture apparatus inan embodiment.

FIG. 2 is a schematic configuration diagram of a microscope unit in thepresent embodiment.

FIG. 3 is a flow chart for explaining an example of an observationoperation with the microscope unit in the present embodiment.

FIG. 4A is a diagram showing an example of a microscope image.

FIG. 4B is a diagram showing a state where a cellular region and anon-cellular region are separated in FIG. 4A.

FIG. 5 is a diagram showing an example of a relationship between theheight of a contour surface of a cultured cell shown in FIG. 4A and afocusing position.

DETAILED DESCRIPTION

A configuration of a cell culture apparatus in an embodiment will beexplained below with reference to the drawings. FIG. 1 is an elevationview of an outline of a cell culture apparatus in the presentembodiment.

A cell culture apparatus 11 in the present embodiment has an uppercasing 12 and a lower casing 13. In an assembled state of the cellculture apparatus 11 shown in FIG. 1, the upper casing 12 is mounted onthe lower casing 13. Meanwhile, the inner space of the upper casing 12and the lower casing 13 is partitioned by a base plate 14 into an upperspace and a lower space. Moreover, at the front of the cell cultureapparatus 11, a door is provided to carry in/out a culture vessel 19 andmechanical equipment and materials (FIG. 1 shows a state where the dooris open and for the sake of simplification, the door is not shownschematically).

Inside the upper casing 12, a temperature-controlled room 15 to culturea cell is formed. The temperature-controlled room 15 has a temperatureadjusting device and a humidity adjusting device and the inside of thetemperature-controlled room 15 is maintained to be an environmentsuitable for culturing a cell (for example, an atmosphere of atemperature of 37° C. and a humidity of 90%) (in FIG. 1, the temperatureadjusting device and the humidity adjusting device are not shownschematically).

Furthermore, in the temperature-controlled room 15, a stocker 16, amicroscope unit 17, and a vessel carrying device 18 are arranged.

The stocker 16 is arranged on the left side (left side in FIG. 1) of thetemperature-controlled room 15 when viewed from the front of the uppercasing 12. The stocker 16 has a plurality of shelves and it is possibleto accommodate a plurality of the culture vessels 19 on each shelf ofthe stoker 16. Then, in each culture vessel 19, cells are placed alongwith the culture media.

The microscope unit 17 is arranged on the right side (right side inFIG. 1) of the temperature-controlled room 15 when viewed from the frontof the upper casing 12. In the microscope unit 17, it is possible tomake a time-lapse observation of a cell in the culture vessel 19.

The microscope unit 17 is fitted to and installed in an opening of thebase plate 14 of the upper casing 12. The microscope unit 17 has asample table 21, a stand arm 22 that extends over the sample table 21,and a main body part 23. While the sample table 21 and the stand arm 22are arranged in the temperature-controlled room 15, the main body part23 is placed in the lower caging 13. With such a configuration, it ismade possible to make an observation of a cell in the culture vessel 19with the environmental conditions unchanged.

Moreover, the sample table 21 is configured by a transparent materialand the culture vessel 19 can be mounted thereon. The sample table 21 isconfigured so as to be capable of moving in the horizontal direction andof adjusting the position of the culture vessel 19 mounted on the topsurface.

The vessel carrying device 18 is arranged in the center of thetemperature-controlled room 15 when viewed from the front of the uppercasing 12. The vessel carrying device 18 is configured by attaching armsfor sandwiching the culture vessel 19 to the tip end of a vertical robothaving articulated arms. Due to this, it is possible for the vesselcarrying device 18 to deliver the culture vessel 19 with the stocker 16and the sample table 21.

Next, the configuration of the microscope unit 17 in the presentembodiment is explained with reference to FIG. 2. The microscope unit 17has the above-mentioned sample table 21, a microscopic optical system(31 to 39), a lens driving section 24, an imaging section 25, a memory26, and a control section 27. Here, the lens driving section 24, thememory 26, and the imaging section 25 are connected with the controlsection 27, respectively.

The microscopic optical system has a light source 31, a collector lens32, a mirror 33, a field lens 34, a ring diaphragm 35 (aperturediaphragm), a condenser lens 36, an objective lens 37, a phase ring 38,and an image forming lens 39. Here, the light source 31, the mirror 33,the field lens 34, the ring diaphragm 35, and the condenser lens 36 arearranged in the stand arm 22. Moreover, the objective lens 37, the phasering 38, and the image forming lens 39 are arranged in the main bodypart 23 (under the sample table 21).

In FIG. 2, the illumination light emitted from the light source 31becomes parallel light by the collector lens 32 and guided downward inFIG. 2 by the mirror 33. Then, the illumination light reflected from themirror 33 is condensed by the field lens 34 and enters the ringdiaphragm 35. The ring diaphragm 35 is a disc having a ring-shapedopening and turns the illumination light into light having passedthrough a ring-shaped diaphragm. The ring diaphragm 35 is arranged inthe focal position on the front side of the condenser lens 36. Moreover,the condenser lens 36 condenses a light flux having passed through thering diaphragm 35. In this way, the culture vessel 19 mounted on thesample table 21 is illuminated from above with the illumination light.

The objective lens 37 transmits the direct light (zero-order light)having passed through the culture vessel 19 and the diffracted lightthat is generated in accordance with a phase matter (a cultured cell) inthe culture vessel 19. The phase ring 38 is arranged in a positionoptically conjugate to the ring diaphragm 35 in the focal point surfaceposition on the rear side of the objective lens 37. The phase ring 38transmits part of the diffracted light that is generated in the culturedcell and produces a delay (or advance) in the transmitted light phase.The image forming lens 39 forms an enlarged image of the cultured cellin the imaging section 25 based on the direct light and the diffractedlight. In such a microscopic optical system, the light having passedthrough the phase ring 38 interferes with the light having passedthrough other than the phase ring 38, to form an image as a differencein brightness on an image forming surface via the image forming lens 39.In this way, it is possible to make a phase-difference observation of acultured cell.

The lens driving section 24 adjusts the focal point of the microscopicoptical system by driving the objective lens 37 in the direction of theoptical axis (in the vertical direction in FIG. 2).

The imaging section 25 generates data of a microscope image by capturingan image formed by the microscopic observation system. The imagingsection 25 has an image sensor, an analog front end that makes a gainadjustment for the output signal of the image sensor and performs A/Dconversion, and an image processing section that performs various kindsof image processing. In FIG. 2, the individual components of the imagingsection 25 are not shown schematically.

The memory 26 is configured by, for example, a nonvolatile storingmedium, such as a flash memory. In the memory 26, data of a microscopeimage is stored. Moreover, in the memory 26, a program executed by thecontrol section 27 is also stored.

The control section 27 is a processor that totally controls each part ofthe microscope unit 17. Further, the control section 27 functions alsoas a region separating section 28, a focusing position calculatingsection 29, and a three dimensional information calculating section 30by executing the program stored in the memory 26. The explanation of theregion separating section 28, the focusing position calculating section29, and the three dimensional information calculating section 30 will begiven later.

An example of the observation operation with the microscope unit 17 inthe present embodiment will be explained below with reference to theflowchart in FIG. 3. In the example of the operation, the microscopeunit 17 calculates the volume of a cultured cell in the field. In theexample in FIG. 3, explanation is given on the premise that the culturevessel 19, which is an observation object, is in the state of beingcarried on the sample table 21 by the vessel carrying device 18.Meanwhile, in the culture vessel 19 shown in FIG. 4A, there are anattached cell and a floating cell in the culture solution.

Step S101: The control section 27 captures a plurality of microscopeimages with different focal positions in the same field range.Specifically, the control section 27 turns on the light source 31 toilluminate the culture vessel 19 and at the same time, captures amicroscope image by driving the imaging section 25. After that, thecontrol section 27 shifts the position of the objective lens 37 by thelens driving section 24 in the direction of optical axis (Z axisdirection) and then repeats capturing a microscope image under the sameshooting conditions. For example, a plurality of microscope images,sectioning of coordinates Z1 to Z5 of which is performed from the bottomtoward the top of the culture vessel 19 is acquired. In this way, aplurality of microscope images with different focal positions in thesame field is generated (refer to FIG. 4B). Here, the data of eachmicroscope image captured in S101 is recorded in the memory 26 by thecontrol section 27. It is assumed that the data of each microscope imageis associated with focal position information indicative of the focalposition of the objective lens 37 when the image is captured.

Step S102: The region separating section 28 finds a variance value ofbrightness of each pixel of the shooting screen corresponding to themicroscope image by using a brightness value of a plurality ofmicroscope images (S101). For example, the region separating section 28calculates each variance value of brightness of all of the pixels of thecoordinates (x, y) of the shooting screen from a plurality of brightnessvalues indicated by the pixel of the coordinate (x, y) of eachmicroscope image.

Step S103: The region separating section 28 separates a cellular regionwhere a cultured cell is located from a non-cellular region where nocultured cell is located, based on the variance value of brightness(S102).

As an example, the region separating section 28 compares the variancevalue of brightness in the coordinates (x, y) of the shooting screenwith a threshold value for determining a region. Here, in the positionwhere a cultured cell is located on the shooting screen, the brightnesschanges between images due to the change in focal position. For thisreason, in the position where a cultured cell is located, the variancevalue of brightness becomes larger. On the other hand, in the positionwhere no cultured cell is located on the shooting screen, the brightnesshardly changes between images even if the focal position changes.Accordingly, in the position where a cultured cell is located, thevariance value of brightness becomes very small.

Consequently, when the variance value of brightness in the coordinates(x, y) is more than or equal to a threshold value, the region separatingsection 28 determines that the pixel in the coordinates (x, y) is in acellular region. On the other hand, when the variance value ofbrightness is less than the threshold value, the region separatingsection 28 determines that the pixel in the coordinates (x, y) is in anon-cellular region. Then, the region separating section 28 separatesthe cellular region from the non-cellular region on the shooting screenby making the above-mentioned determination in each coordinate of theshooting screen. The state where the cellular region is separated fromthe non-cellular region on the shooting screen is shown schematically inFIG. 4B.

Additionally, in S103, the region separating section 28 performsgrouping processing to group the pixels in the cellular region andlabeling processing to associate each grouped cellular region withidentification information (for example, identification number etc.) onone-on-one level.

Step S104: Further, the region separating section 28 sets a brightnessthreshold value by using the brightness value in the non-cellular region(S103). As an example, the region separating section 28 selects anarbitrary frame from among a plurality of microscope images (S101).Then, the region separating section 28 finds the above-mentionedbrightness threshold value by averaging the brightness value of eachpixel in the non-cellular region in the selected frame. The brightnessthreshold value found in S104 is used by the focusing positioncalculating section 29 when finding the focusing position in each pixelin the cellular region.

Step S105: The focusing position calculating section 29 finds thefocusing position in each pixel in the cellular region (S103) of theshooting screen. This focusing position indicates the contour of a cell.That is, when a phase difference image (microscope image) is used, thereis a characteristic that the larger the change in phase, the larger thebrightness of the phase difference becomes in the region. For thisreason, in general, the contour region of the cell has the maximumbrightness. As an example, the focusing position calculating section 29in S105 performs the following processing (1) to (3).

(1) The focusing position calculating section 29 selects a target pixel,which is a processing object, from among those in the cellular region ofthe shooting screen. Then, the focusing position calculating section 29acquires the brightness value corresponding to the above-mentionedtarget pixel from the data (S101) of each microscope image.

(2) The focusing position calculating section 29 compares eachbrightness value acquired in the above-mentioned (1) with the brightnessthreshold value (S104). Here, in a pixel in which the change inbrightness due to a phase difference appears in the direction ofbrightness, the relative brightness value is the largest when theobjective lens 37 is in the focusing position. On the other hand, in apixel in which the change in brightness due to a phase differenceappears in the direction of darkness, the brightness value is relativelythe smallest when the objective lens 37 is in the focusing position.

For this reason, when each brightness value acquired in theabove-mentioned (1) is more than or equal to the brightness thresholdvalue, the focusing position calculating section 29 performs thefollowing processing. In this case, the focusing position calculatingsection 29 finds the maximum point of the brightness value in thespatial direction by using the brightness value acquired in theabove-mentioned (1). Then, the focusing position calculating section 29determines that the focal position of the objective lens 37 where themicroscope image indicating the above-mentioned maximum point can beacquired is the focusing position in the target pixel. In this way, itis possible to find the focusing position in a pixel in which the changein brightness due to a phase difference appears in the direction ofbrightness.

On the other hand, when each brightness value acquired in theabove-mentioned (1) is less than the brightness threshold value, thefocusing position calculating section 29 performs the followingprocessing. In this case, the focusing position calculating section 29finds the minimum point of the brightness value in the spatial directionby using the brightness value acquired in the above-mentioned (1). Then,the focusing position calculating section 29 determines that the focalposition of the objective lens 37 where the microscope image indicatingthe above-mentioned minimum point can be acquired is the focusingposition in the target pixel. For this reason, it is possible to findthe focusing position in a pixel in which the change in brightness dueto a phase difference appears in the direction of darkness.

Meanwhile, in this (2), the focusing position calculating section 29 mayextract a microscope image the brightness value of which is the maximum(or minimum) in the position of the target pixel and at the same time,may determine that the focal position of the objective lens 37corresponding to the extracted microscope image is the focusingposition. Alternatively, the focusing position calculating section 29may find an interpolation curve that interpolates each brightness valuein the position of the target pixel and at the same time, may find thefocusing position of the objective lens 37 by calculation based on themaximum point (or minimum point) on the interpolation curve.

(3) The focusing position calculating section 29 changes the targetimage, which is a processing object, to another pixel, repeats theprocessing in the above-mentioned (2), and finds the focusing positionof the objective lens 37 in each pixel in the cellular region. Then, thefocusing position calculating section 29 generates a focusing positionmap indicating the focusing position of the objective lens 37 for eachpixel in the cellular region of the shooting screen.

Step S106: The three dimensional information calculating section 30finds the height of the contour surface of the cultured cell (theposition of the contour surface of the cultured cell in the direction ofoptical axis) based on the focusing position of the objective lens 37.That is, the three dimensional information calculating section 30 findsthe height of the contour surface of the cultured cell corresponding tothe position of the pixel from the focusing position (S105) of theobjective lens 37 in each pixel by using the publicly-known opticalfundamental expression (Gaussian formula of lens). In this way, it ispossible for the three dimensional information calculating section 30 toobtain data of a three dimensional distance image (point group data)indicating the height of the contour surface of the cultured cell. Arelationship between the height of the contour surface of the culturedcell and the focusing position in the example in FIG. 4A is shown inFIG. 5.

Step S107: The three dimensional information calculating section 30determines which kind of a floating cell and an attached cell eachcellular region grouped in S102 belongs to, based on the height of thecontour surface of the cultured cell (S106).

Specifically, first, the three dimensional information calculatingsection 30 acquires the bottom position of the culture vessel 19. Forexample, the three dimensional information calculating section 30regards the position where the height of the contour surface is thelowest as the bottom position of the culture vessel 19 in all of thecellular regions in the shooting screen (refer to FIG. 5). This isbecause the position of the contour surface corresponding to theabove-mentioned condition can be thought to correspond to the outer edgeof the attached cell attached to the bottom of the culture vessel 19.

Then, the three dimensional information calculating section 30determines a cell, the contour surface height of which is a fixeddistance (threshold value h) or more apart from the bottom position ofthe culture vessel 19 of the group of the cellular region, as a floatingcell (refer to FIG. 5). In this way, it becomes possible for the threedimensional information calculating section 30 to automatically identifya floating cell and an attached cell in the shooting screen.

Step S108: The three dimensional information calculating section 30estimates a total volume of attached cells included in the shootingscreen. Specifically, first, the three dimensional informationcalculating section 30 excludes from processing objects those determinedas a floating cell in step S107 in the cellular region of the shootingscreen. Next, the three dimensional information calculating section 30finds the area of a unit pixel by taking into account the magnificationof the objective lens 37. Then, the three dimensional informationcalculating section 30 estimates the volume of each cultured cell byintegrating the height (S106) of each pixel included in the cellularregion (attached cell), which is a processing object. After that, thethree dimensional information calculating section 30 generates data ofthe total volume of the attached cells included in the shooting screenand records it in the memory 26. With that, the explanation of theflowchart in FIG. 3 is completed.

The action and effect of the present embodiment will be explained below.The microscope unit 17 in the present embodiment finds each focusingposition of the pixel in the cellular region by using a plurality ofphase-difference observation images with different focal positions andacquires a three dimensional shape of the cultured cell based on thefocusing position. Therefore, it is possible to acquire threedimensional information of the cultured cell without damaging the cellby the radiation of excited light or dyeing with the microscope unit 17in the present embodiment. Further, with the microscope unit 17 in thepresent embodiment, the cultured cell is observed in thetemperature-controlled room 15, and therefore, it is unlikely that thecultured cell is damaged by the change in the environmental conditionsat the time of observation.

Moreover, the microscope unit 17 in the present embodiment finds threedimensional information of a cultured cell by using a plurality ofphase-difference observation images after narrowing the cellular regionin advance in the shooting screen. In this way, it is possible toalleviate the load of calculation when finding three dimensionalinformation of the cultured cell with the microscope unit 17 in thepresent embodiment.

Further, with the microscope unit 17 in the present embodiment, it ispossible to distinguish the floating cell from the attached cell and toestimate the volume of the cultured cell based on the three dimensionalinformation of the cultured cell, and therefore, it is possible tofurther improve the functionality of the cell culture apparatus 11.

In particular, when the time-lapse observation accompanied by a seriesof processes shown in FIG. 3 is made by using the cell culture apparatus11 in the present embodiment, a growth curve can be obtained, whichindicates the transition of the total volume of the attached cells overtime. In this way, with the cell culture apparatus 11 in the presentembodiment, it also becomes possible to accurately observe, for example,laminated cells, and evaluate the cultured state thereof.

Supplementary of the Embodiment

(1) It may also be possible for the region separating section 28 in theabove-mentioned embodiment to perform region separation only with thebrightness value without using the variance value. Meanwhile, when theregion separating section 28 performs region separation by using thevariance value as in the above-mentioned embodiment, there is a meritthat the contrast becomes stronger and it becomes easier to performregion separation.

(2) The microscope unit 17 in the above-mentioned embodiment may be adifferential interference microscope that has a birefringence opticalmaterial on the focal surface on the side of the light source and on thefocal surface on the side of the image and which forms an image byconverting a phase difference of a sample into a brightness differenceby interference of light waves (the optical system of a differentialinterference microscope is publicly known, and therefore, its detailedexplanation is omitted and not shown schematically).

(3) The operation of the microscope unit 17 in the above-mentionedembodiment is merely an example and the processing shown in FIG. 3 maybe combined appropriately and performed. As an example, the microscopeunit 17 may omit the operation in S107 and find the volume of all thecells included in the shooting screen. Alternatively, it may beconfigured such that the microscope unit 17 performs only the processingto distinguish the floating cell from the attached cell. That is, themicroscope unit 17 may complete the series of processes withoutperforming the processing in S108 after the operation in S107.

(4) The configuration of the microscope unit 17 in the above-mentionedembodiment is merely an example. For example, the microscope unit 17 ofthe present invention may have a configuration in which the light source31 is arranged under the sample table 21 and on the other hand, theobjective lens 37, or the like is arranged above the culture vessel 19.Further, the microscope apparatus of the present invention is notlimited to that arranged in the temperature-controlled room 15 and it isobvious that a microscope apparatus used outside thetemperature-controlled room 15 is also included in the technical scopeof the present invention.

(5) It may be configured such that the microscope unit 17 in theabove-mentioned embodiment adjusts the focal point by driving theposition of the sample table 21, instead of the objective lens 37, inthe direction of optical axis.

(6) The algorithm to find the volume of the cultured cell is not limitedto the example in the above-mentioned embodiment. As an example, it maybe configured such that the three dimensional information calculatingsection 30 approximately calculates the volume of each grouped culturedcell by approximating the area of the cellular region with other shapes,such as a rectangle and a circle, and multiplying the height at anarbitrary position of the cellular region (or the average height in thecellular region).

(7) In the above-mentioned embodiment, although the example in whicheach function of the region separating section 28, the focusing positioncalculating section 29, and the three dimensional informationcalculating section 30 is realized by software, that is, programs, isexplained, it may also be possible to realize these components byhardware by using ASIC.

(8) Meanwhile, the programs stored in the memory 26 in theabove-mentioned embodiment may be firmware programs that are updated byupgrading etc. That is, it may also be possible to provide the functionsof the microscope apparatus of the present invention by updating thefirmware programs of an already existing microscope apparatus.

The many features and advantages of the embodiment are apparent from thedetailed specification and, thus, it is intended by the appended claimedto cover all such features and advantages of the embodiment that fallwithin the true spirit and scope thereof. Further, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the inventive embodiment to the exactconstruction and operation illustrated and described, and accordinglyall suitable modifications and equivalents may be resorted to, fallingwithin the scope thereof.

What is claimed is:
 1. A microscope apparatus comprising: a microscopicoptical system; an imaging section capturing, along an optical axis ofthe microscopic optical system, a plurality of images at different focalpositions, the plurality of images including a cell; a calculatingsection calculating height information of the cell in a direction of theoptical axis of the microscopic optical system based on the differentfocal positions and a brightness value of pixels in the same position ofthe plurality of images in the direction of the optical axis, thecalculating section finding a focusing position of the cell from a pixelhaving the maximum or minimum brightness value among the pixels in thesame position of the plurality of images, and generating a height of acontour surface of the cell as the height information by using the foundfocusing position of the cell; and a determining section determiningwhether the cell is a floating cell or an attached cell based on theheight information, the determining section determining a cell havingequal or higher contour surface height than a threshold value as thefloating cell, and determining a cell having a shorter contour surfaceheight than the threshold value as the attached cell.
 2. The microscopeapparatus according to claim 1, wherein the threshold value is a fixeddistance apart from a basic height which is the lowest height of thecontour surface.
 3. The microscope apparatus according to claim 1,wherein the calculating section calculates a volume of the attached cellby using three dimensional information including the height information.4. The microscope apparatus according to claim 1, wherein the imagingsection captures the plurality of images in the same field by adjustinga distance between the cell and a focal position of the microscopicoptical system in the direction of the optical axis.
 5. The microscopeapparatus according to claim 1, wherein: the microscopic optical systemis capable of performing a phase-difference observation of the cell; andthe plurality of images include a phase difference image.
 6. Themicroscope apparatus according to claim 1, wherein: the microscopicoptical system is capable of performing a differential interferenceobservation of the cell; and the plurality of images include adifferential interference image.
 7. A method for acquiring heightinformation of a cell comprising: capturing, along an optical axis ofthe microscopic optical system, a plurality of images at different focalpositions, the plurality of images including a cell; calculating heightinformation of the cell in a direction of the optical axis of themicroscopic optical system based on the different focal positions and abrightness value of pixels in the same position of the plurality ofimages in the direction of the optical axis the calculating including:finding a focusing position of the cell from a pixel having the maximumor minimum brightness value among the pixels in the same position of theplurality of images; and generating a height of a contour surface of thecell as the height information by using the found focusing position ofthe cell; and determining whether the cell is a floating cell or anattached cell based on the height information, the determining includingdetermining a cell having equal or higher contour surface height than athreshold value as the floating cell, and determining a cell having alower contour surface height than the threshold value as the attachedcell.
 8. The method according to claim 7, wherein the threshold value isa fixed distance apart from a basic height which is the lowest height ofthe contour surface.
 9. The method according to claim 7, furthercomprising calculating a volume of the attached cell by using threedimensional information indicating the height information.
 10. Themethod according to claim 7, further comprising capturing the pluralityof images in the same field by adjusting a distance between the cell anda focal position of the microscopic optical system in the direction ofthe optical axis.
 11. The method according to claim 7, wherein: themicroscopic optical system is capable of performing a phase-differenceobservation of the cell; and the plurality of images include a phasedifference image.
 12. The method according to claim 7, wherein: themicroscopic optical system is capable of performing a differentialinterference observation of the cell; and the plurality of imagesinclude a differential interference image.
 13. A non-transitory computerreadable storage medium to store a program causing a computer toexecute: capturing, along an optical axis of the microscopic opticalsystem, a plurality of images at different focal positions, theplurality of images including a cell; calculating height information ofthe cell in a direction of the optical axis of the microscopic opticalsystem based on the different focal positions and a brightness value ofpixels in the same position of the plurality of images in the directionof the optical the calculating including: finding a focusing position ofthe cell from a pixel having the maximum or minimum brightness valueamong the pixels in the same position of the plurality of images; andgenerating a height of a contour surface of the cell as the heightinformation by using the found focusing position of the cell; anddetermining whether the cell is a floating cell or an attached cellbased on the height information, the determining including determining acell having equal or higher contour surface height than a thresholdvalue as the floating cell, and determining a cell having a lowercontour surface height than the threshold value as the attached cell.